A critical feature of a polypeptide is its ability to fold into a three dimensional conformation or structure. Polypeptides usually have a unique conformation which, in turn, determines their function. The conformation of a polypeptide has several levels of structure. The primary structure is a linear sequence of a series of amino acids linked into a polypeptide chain. The secondary structure describes the path that the polypeptide backbone of the polypeptide follows in space, and the tertiary structure describes the three dimensional organization of all the atoms in the polypeptide chain, including the side groups as well as the polypeptide backbone.
Covalent and noncovalent interactions between amino acids determine the conformation of a polypeptide. The most common covalent bond used in establishing the secondary and tertiary structure of a polypeptide is the formation of disulfide bridges between two cysteine residues (forming cystine). The formation of noncovalent bonds is influenced by the aqueous environment such as water. A large number of noncovalent interactions, such as van der Waals, ionic, hydrophobic and hydrogen-bonded interactions, contribute to the way in which a polypeptide folds. Hydrophobic interactions, which occur between amino acids with nonpolar side chains, are particularly important because they associate to exclude water. These side chains generally form the core of the polypeptide, where they are mostly inaccessible to water.
The secondary structure of polypeptides can be divided into two general classes: .alpha.-helix and .beta.-sheet. An .alpha.-helix is stabilized by hydrogen bonding and side chain interactions between amino acids three and four residues apart in the same polypeptide chain, whereas a .beta.-sheet is stabilized by hydrogen bonding and side chain interactions between amino acids more distant in a polypeptide chain and in different polypeptide chains. A complete understanding of the construction of .alpha. helices and .beta. sheets is important for the manipulation of the structure and function of polypeptides.
A major challenge in de novo polypeptide design (more often referred to as de novo peptide design), which is the design of polypeptides (or peptides) from scratch, is the engineering of a polypeptide having the folding stability of the native structure of a natural polypeptide. Several polypeptides have been designed with the .alpha. helix as the major structural element. Few polypeptideshave been designed with the .beta. sheet as the major structural element. Unlike .alpha. helices where there is a regular succession of hydrogen bonds between amides three and four residues apart in the sequence, .beta. sheets are formed by residues at variable and often distant positions in the sequence. In addition, they tend to form aggregates in solution and precipitate under physiological conditions. A major difficulty in designing a structurally stable .beta. polypeptide is dealing with the interactions between .beta. sheets.
Designing a polypeptide to form a .beta.-sheet has in the past usually been based on one of a number of structural propensity scales known in the art. These scales are derived either statistically from structural databases of known folded polypeptides or by making single or minimal site-specific changes in a fully folded polypeptide. See, for example, C. A. Kim, et al., Nature, 362, 267 (1993); D. L. Minor, et al., Nature, 371, 264 (1994); D. L. Minor, et al., Nature, 367, 660 (1994); and C. K. Smith, et al., Biochemistry, 33, 5510 (1994). However, such scales are generally less useful when designing de novo .beta.-sheet folds in short peptides where considerably more .beta.-sheet and/or side-chain surface (particularly hydrophobic surface) will be exposed to water. D. E. Otzen, et al., Biochemistry, 34, 5718 (1995).
Betabellin was one of the first de novo designed class of .beta.-sheet peptides. J. Richardson, et al., Biophys. J., 63, 1186 (1992). It was intended to fold into a sandwich of two identical four-stranded, antiparallel .beta. sheets. A more recent version of betabellin, betabellin 14D, was designed by Yan, et al., Protein Science, 3, 1069, (1994). Quinn, et al. designed betadoublet, which is similar to betabellin but contains only naturally encoded amino acids. T. P. Quinn, et al., Proc. Natl. Acad. Sci. U.S.A., 91, 8747 (1994).
However, peptides in the betabellin and betadoublet series show limited solubility in water and minimal, highly transient .beta.-sheet structure, i.e., nonstable structures. The best betabellin made thus far, Betabellin peptide 14D, for example, becomes less soluble at pH values above 5.5 making it impractical for use at a physiological pH. Moreover the .beta.-sheet structure formed by peptide 14D relies on the presence of an intermolecular disulfide bridge to yield a dimeric species. The peptides of the present invention do not have these limitations. Betadoublet, which has the same predicted antiparallel .beta.-sheet motif as betabellin, is even less water soluble, and only at a lower pH of about 4, and fails to show any compact, stable folding, i.e., structure.
Water solubility and pH ranges are important to peptide function. A polypeptide that is not soluble under physiological conditions (i.e., in water at a pH of about 7.0-7.4 and in about 150 mM NaCl or an equivalent physiological salt) is not functional and is therefore not useful. Neither the betabellin nor the betadoublet strategies for peptide design achieved sufficient solubility, peptide compactness, or peptide self-association under physiological conditions.
Hence, there remains a need for .beta.-sheet forming peptides that are not only water soluble, but soluble at physiological conditions, and self associate.